• Journal of Microbiology and Biotechnology
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• v.28 no.4
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• pp.571-578
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• 2018

Biofuel production using lignocellulosic biomass is gaining attention because it can be substituted for fossil fuels without competing with edible resources. However, because Saccharomyces cerevisiae does not have a ${\text\tiny{D}}$-xylose metabolic pathway, oxidoreductase or isomerase pathways must be introduced to utilize ${\text\tiny{D}}$-xylose from lignocellulosic biomass in S. cerevisiae. To elucidate the biochemical properties of xylose isomerase (XI) from Piromyces sp. E2 (PsXI), we determine its crystal structure in complex with substrate mimic glycerol. An amino-acid sequence comparison with other reported XIs and relative activity measurements using five kinds of divalent metal ions confirmed that PsXI belongs to class II XIs. Moreover kinetic analysis of PsXI was also performed using $Mn^{2+}$, the preferred divalent metal ion for PsXI. In addition, the substrate-binding mode of PsXI could be predicted with the substrate mimic glycerol bound to the active site. These studies may provide structural information to enhance ${\text\tiny{D}}$-xylose utilization for biofuel production.

• KSBB Journal
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• v.19 no.3
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• pp.226-230
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• 2004

Fermentation characteristics of D-xylose into xylitol by Candida mogii ATCC 18364, a potential xylitol producer from rice straw hemicellulose hydrolyzates, were investigated. The influences of the most important operational variables on xylitol production were examined. The best results in xylitol production were obtained in shake-flask fermentations when 3.0 g/L initial cell concentration of 12 hr-old cells grown in D-glucose containing medium were used as inoculum. The oxygen availability is a critical factor in xylose fermentation, therefore, xylose conversion into xylitol was investigated in a 2-L fermenter at different stirring rates. Maximum xylitol production was obtained with an aeration rate of 1 vvm at a stirring rate of 200 rpm.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.19 no.3
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• pp.212-218
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• 1986

In order to isolate antioxigenic substrates, the browning reaction products of xylose and various amino acids were analysed by TLC and dialysis. Rf values of browning reaction products of xylose and hydrophobic amino acids separated on silica gel TLC plate were shown in the range of 0.38 to 0.56 and that of basic amino acids was around 0.2. Browning reaction products made from xylose and Trp were separated on TLC into four bands with Rf values of 0.25, 0.55, 0.81 and 0.91 respectively. Among these the bands with Rf values of 0.25 and 0.55 appeared having strong antioxidant activity. The band of Rf 0.55 which showed the highest activity was positive to Prochazka reagent and had an absorption maximum at 275 nm. In dialysis of the xylose-Trp browning reaction products, the undialysed fraction (inner solution) was repsponsible to the antioxidant activity, which was separated into two bands with Rf values of 0.25 and 0.55 on TLC. The inner fractions of the browning products of xylose and His or Arg were also apparent in antioxdant activity.

• Journal of Korea Technical Association of The Pulp and Paper Industry
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• v.44 no.3
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• pp.9-14
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• 2012

Formed fermentation inhibitors during acid saccharification leads to poor alcohol production based on lignocellulosic bio-alcohol production process. In this work, it is focused on the formation of fermentation inhibitors from xylan, which is influenced by reaction tempearature and time of acidic sacharifiaction of xylose and glucuronic acid. In second step of concentrated acid hydrolysis, part of xylose and glucuronic acid was converted to furfuraldehyde and formic acid by dehydration and rearrangement reactions. Furfural was form from xylose, which was highly sensitive to reaction temperature. Formic acid was come from both xylose and glucuronic acid, which supposed to main inhibitor in biobutanol fermentation. Reaction temperature of second hydrolysis was main variables to control the furfural and formic acid generation. Careful control of acid saccharification can reduce generation of harmful inhibitors, especially second step of concentrated sulfuric acid hydrolysis process.

• Journal of Microbiology and Biotechnology
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• v.23 no.4
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• pp.560-564
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• 2013

${\small{D}}$-Ribose is a value-added five-carbon sugar used for riboflavin production. To investigate the effects of oxygen supply and mixed sugar concentration on microbial production of ${\small{D}}$-ribose, a transketolase-deficient Bacillus subtilis SPK1 was cultured batch-wise using xylose and glucose. A change of agitation speed from 300 rpm to 600 rpm at 1 vvm of air supply increased both the xylose consumption rate and ${\small{D}}$-ribose production rate. Because the sum of the specific consumption rates for xylose and glucose was similar at all agitation speeds, metabolic preferences between xylose and glucose might depend on oxygen supply. Although B. subtilis SPK1 can take up xylose and glucose by the active transport mechanism, a high initial concentration of xylose and glucose was not beneficial for high ${\small{D}}$-ribose production.

• Current Research on Agriculture and Life Sciences
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• v.32 no.4
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• pp.199-202
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• 2014

The bioconversion of cellulosic biomass hydrolyzates consisting mainly of glucose and xylose requires the use of engineered Saccharomyces cerevisiae expressing a heterologous xylose pathway. However, there is concern that a fungal xylose pathway consisting of NADPH-specific xylose reductase (XR) and $NAD^+$-specific xylitol dehydrogenase (XDH) may result in a cellular redox imbalance. However, the glycerol biosynthesis and glycerol degradation pathways of S. cerevisiae, termed here as the glycerol cycle, has the potential to balance the cofactor requirements for xylose metabolism, as it produces NADPH by consuming NADH at the expense of one mole of ATP. Therefore, this study tested if the glycerol cycle could improve the xylose metabolism of engineered S. cerevisiae by cofactor balancing, as predicted by an in-silico analysis using elementary flux mode (EFM). When the GPD1 gene, the first step of the glycerol cycle, was overexpressed in the XR/XDH-expressing S. cerevisiae, the glycerol production significantly increased, while the xylitol and ethanol yields became negligible. The reduced xylitol yield suggests that enough $NAD^+$ was supplied for XDH by the glycerol cycle. However, the GPD1 overexpression completely shifted the carbon flux from ethanol to glycerol. Thus, moderate expression of GPD1 may be necessary to achieve improved ethanol production through the cofactor balancing.

• Journal of Microbiology and Biotechnology
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• v.30 no.12
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• pp.1944-1949
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• 2020

Mutant sugar transporter ScGAL2-N376F was overexpressed in Kluyveromyces marxianus for efficient utilization of xylose, which is one of the main components of cellulosic biomass. K. marxianus ScGal2_N376F, the ScGAL2-N376F-overexpressing strain, exhibited 47.04 g/l of xylose consumption and 26.55 g/l of xylitol production, as compared to the parental strain (24.68 g/l and 7.03 g/l, respectively) when xylose was used as the sole carbon source. When a mixture of glucose and xylose was used as the carbon source, xylose consumption and xylitol production rates were improved by 195% and 360%, respectively, by K. marxianus ScGal2_N376F. Moreover, the glucose consumption rate was improved by 27% as compared to that in the parental strain. Overexpression of both wild-type ScGAL2 and mutant ScGAL2-N376F showed 48% and 52% enhanced sugar consumption and ethanol production rates, respectively, when a mixture of glucose and galactose was used as the carbon source, which is the main component of marine biomass. As shown in this study, ScGAL2-N376F overexpression can be applied for the efficient production of biofuels or biochemicals from cellulosic or marine biomass.

• Journal of Applied Biological Chemistry
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• v.53 no.1
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• pp.1-7
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• 2010

xylA promoter is a major promoter in xylose operon of Escherichia coli. xylA promoter is sufficient as the promoter for the construction of new expression vector because this promoter was tightly controlled and induced by the addition of xylose. For the construction of xylose-inducible expression vector, 600 bp of xylA promoter was ligated between AatII and HindIII of pUC18, named pXA600. In order to investigate the effect of XylR protein encoded by xylR gene on the xylA promoter, 1,988 bp of xylR gene including its promoter was ligated into downstream of multiple cloning site to the opposite direction of xylA promoter in pXA600, named pXAR600. For the measurement of expression level, 3,048 bp of lacZ structural gene was fused into xylA promoter in both plasmids pXA600 and pXAR600 as a reporter gene, named pXA600-lacZ and pXAR600-lacZ, respectively. The $\beta$-galactosidase activity of pXA600-lacZ and pXAR600-lacZ in E. coli JM109 was determined to be 1,641 and 2,304 unit by the induction with xylose in LB medium, respectively. The $\beta$-galactosidase activity of pXAR600-lacZ/JM109 was about 1.4 times higher by the induction with xylose than that of pXA600-lacZ/JM109. The $\beta$-galactosidase activity of pXA600-lacZ and pXAR600-lacZ in E.coli JM109 showed 6,282 and 9,320 unit by the induction with xylose in DM minimal medium, respectively. A regulator, xylR protein works as an activator for the gene expression by the addition of xylose in the xylose-inducible vectors because the level of gene expression in pXA600 is increased by the insertion of xylR gene into the same vector. The xynA gene of Streptomyces thermocyaneoviolaceus cloned in pXA600 and pXAR600 was successfully expressed in E. coli BLR(DE3). As a result, plasmids pXA600 and pXAR600 using xylA promoter are sufficient as new expression system to produce a foreign protein in E. coli.

• Applied Biological Chemistry
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• v.39 no.6
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• pp.430-436
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• 1996

The induction by xylose and repression by glucose of xylose isomerase(XI) were investigated to elucidate the regulation for production of XI in Escherichia coli. Regulation for expression of xyIA gene which codes XI is under control of xylR which is a regulatory gene for xylose catabolism. When xyIR gene was resided in chromosome, the inductions of XI by the addition of 0.4% xylose were increased to 1.9 and 1.7-fold in case of locating on multicopy(pEX202/DH77) and low copy Plasmid(pEX102/DH77), respectively, as compared with that of xylA gene which was resided in chromosome(JM109). xyIR gene product derived from xyIR gene on chromosome might react to xylA gene on the plasmid as same as xylA gene on chromosome. In JM109 and xylA transformant; pEX202/DH77 and pEX102/DH77, the inductions of XI were completely repressed by the addition of 0.2% glucose and these catabolite repressions were derepressed by the addition of 1 mM cAMP In comparison with the addition of 0.4% xylose only for the induction XI was inductively produced 1.7 to 2-fold with the addition of xylose plus 1 mM cAMP in DM minimal media. pEX13/TP2010, xylA transformant of the deficient mutant($xyl^-,\;cya^-$; TP2010) of XI and cAMP production, did not induce XI by the addition of xylose only but induced in case of simultaneous addition of xylose and cAMP. These results show that cAMP and xylose are the indispensable effectors for the induction and derepression of Xl in E. coli.

• Current Research on Agriculture and Life Sciences
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• v.10
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• pp.125-135
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• 1992

Nine strains of xyl mutants that could not utilize xylose as a carbon source were isolated from E. coli JM109 by the treatment of NTG in order to investigate the regulation of xylose operon and to use recipient cells for the cloning of xylR gene. For the characterization of all isolated mutants, colony colors of all mutants on MacConkey-xylose and MacConkey-xylulose agar plate were observed for the utilization of xylose and xylulose, and the growth level and the activity of xylose isomerase and xylulokinase were determined in need. The isolated xylR/T mutants formed the white colony on MacConkey-xy-lose and MacConkey-xylulose agar plate. They did not detect the activity of xylose isomerase, and the activity of xylose isomerase was not restored in transformants of xylR/T mutant with pEX13 which contained xylA gene. xylR and xylT mutants were classified from xylR/T mutants depending upon the growth level in minimal medium. xylT mutants; DH13, DH121 and DH125 could grow a little in that medium, but xylR mutants; DH10, DH53, and DH60 could not grow that medium.